The relationship between position and chemical composition is a fundamental issue in the study of biologic systems. For example, position-composition relationiships are important in understanding mosaicism, the clonal evolution of cancer, tissue methylation patterns, cell-cell communication, tissue-specific gene expression, developmental gene expression and cell-specific infection (e.g., viral). At the subcellular level, an understanding of such relationships is critical for gaining insight into the mechanisms by which how chromosomes and other molecules form the architecture of the cell itself. Numerous in situ methods are presently available to study position-composition relationships, including immunocytochemistry (i.e., using antibodies to detect antigen compositions), fluorescence in situ hybridization (i.e., hybridization of nucleic acid probes to detect nucleic acid compositions) and in situ PCR (i.e., locally amplifying a target sequence followed by hybridization with labeled probes). In situ methods detect position-composition relationships by directly contacting biologic material, usually while on a glass slide, with an assay mixture for a particular composition. The in situ assay mixture produces a visible product which co-localizes with the composition it detects, revealing position-composition relationships by visual inspection. Currently available methods have significant limitations including the types of compositions that can be detected, the sensitivity and specificity of detection, the number of assays that can be performed on the same biologic material, the need for significant human interpretation, and the difficulty in automating process steps.
In contrast, analytic methods detect one or more compositions with an assay whose products homogeneously distribute in the assay mixture. The average content of all compositions present in the sample is detected rather than specific position-composition relationships. Despite this drawback, the advantages of analytic methods are significant including a wide variety of detectable compositions, outstanding sensitivity and specificity, multiple and often simultaneous assays on the same material, and the ease by which process and interpretation steps may be automated.
To facilitate the study of biological systems, methods that detect position-composition relationships using the advantages of analytic methods are desirable. A major obstacle to the development of such methods is the need to precisely isolate material in positions of interest from the vast majority of remaining biologic material. In attempts to overcome this obstacle, several physical dissection methods have been described including gross dissection of frozen tissue blocks to enrich for specific cell populations (see Fearon et al., Science 238:193, 1987 and Radford et al., Cancer Res. 53:2947, 1993), and "touch preparations" of frozen tissue specimens (see Kovach et al., J. Natl. Cancer Inst. 83:1004, 1991). These methods do not visualize regions of interest microscopically. Selection capabilities are therefore crude, resulting in significant sample contamination. Microdissection using a dissecting microscope has been described providing for better isolation of selected material (see Emmert-Buck et al., Am. J. Pathol. 145:1285, 1994; Zhuang et al., Am. J. Pathol. 146:620, 1995 and Noguchi et al., Cancer Res. 54:1849, 1994). However, microdissection is highly labor intensive and difficult to automate.
Others have attempted to define position-composition relationships in neoplastic cells by growing xenografts, free of infiltrating nonneoplastic cells, in athymic nude mice (see Schutke et al., Proc. Natl. Acad. Sci. USA 92:5950, 1995 and Caldas et al., Nature Genet. 8:27, 1994). However, this method results in xenograft contamination with up to 50% nonneoplastic murine cells, requires weeks for growth, cannot be automated, applies only to autonomously growing cells, and may artifactually alter the composition of cells through the introduction of mutations in the growing xenograft.
An ultraviolet light-mediated method has been described which destroys unwanted genetic material with ultraviolet irradiation except where it has been manually ink-stained with an ink-pen (see Shibata et al., Am. J. Pathol. 141:539, 1992). This method, known as selective ultraviolet radiation fractionation (i.e., SURF), suffers from crude resolution dictated by the size of the ink-pen and requires up to 30 minutes of ultraviolet exposure to destroy unwanted material. Biomolecules are also limited to those sensitive to ultraviolet light. Other disadvantages include the need to transfer the material to a tube (either with or without the underlying substrate), the manual application of ink, the difficulty of automating process steps, and a mixture typically containing irradiated material in vast abundance. The latter can contribute to inhibiting various analytic methods including sensitive amplification methods such as PCR.
Laser capture microdissection (LCM) is a recently described method that utilizes a transparent thermoplastic film (ethylene vinyl acetate, EVA) to capture cells from glass slides under direct visualization (see Emmert-Buck et al., Science 274:998, 1996). EVA is applied to the surface of a tissue section placed on a glass slide and irradiated with a carbon dioxide laser using infrared radiation. The laser energy is absorbed by the film which adheres to the underlying selected cells, which are selectively procured when the film is removed. Drawbacks of LCM include less than 100% transfer of cells to the EVA film, a glass surface that must be specially prepared so as to facilitate tissue lift-off, and a ragged border between irradiated and non-irradiated regions (i.e., low contrast). Because the entire EVA film is transferred from the slide surface to a reaction tube, there is the potential for contamination of the non-irradiated EVA with tissue fragments. The latter is a significant disadvantage when sensitive amplification methods for detection are employed (e.g., PCR). Also, cumbersome automation and robotics are required in the transfer process resulting in a high system cost. Another disadvantage is a resolution limited by the wavelength of the infrared laser (.about.10 microns) which precludes microdissection of subcellular components.
It has long been known to immobilize active biomolecules into thin films of poly (vinyl) pyrrolidone (PVP), PVA and protein (see Hanazato, Anal. Chim. Acta 193:87, 1987 describing the use of negative photoresists for photopatterning thin films containing biomolecules for the purpose of producing biosensors), Takatsu and Moriizumi, Sensor and Actuators 11:309, 1987; Moriizumi and Miyahara, Sensors and Actuators 7:1, 1985 and Ichimura, U.S. Pat. No 4,272,620 (describing the incorporation of enzymes into the photosensitive mixtures consisting of poly(vinyl alcohol) (PVA) and styrylpyridinium or stilbazolium salt); and Cozzette, U.S. Pat. No. 5,200,051 (describing a photoformable proteinaceous matrix that behaves as a negative photoresist). However, it has been believed that biologic material is relatively labile and therefore incompatible with the direct application of photoresist because such processing commonly includes exposure to organic chemicals, strong acids, strong bases ultraviolet light, and reactive chemical species in the photoresist as generated by light (see Cozzette, U.S. Pat. Nos. 5,200,051 and 5,466,575).
Accordingly, there is a need in the art for improved methods for regional analysis of biologic materials. In particular, methods are needed to detect position-composition relationships using analytic methods. The present invention fulfills these needs and further provides other related advantages.